[unreadable] The homohexameric N-ethylmaleimide Sensitive Factor (NSF) is a key element in most physiologically important vesicular trafficking and exocytosis events (i.e. neurotransmission, hemostasis, immune responses). As an AAA ATPase, NSF acts as a "protein helicase" unwinding spent SNARE (SNAP receptor) complexes after they have mediated membrane fusion. Dysfunction of NSF leads to stark phenotypes such as paralysis or growth arrest. NSF protomers have three contiguous domains (NSF-N, NSF-D1, and NSF-D2); each contributes uniquely to activity. Despite our advances, little is known about which structural elements are required for NSF to bind its adaptor protein, alpha SNAP (Soluble NSF Attachment Proteins), and to use the chemical energy from ATP hydrolysis to disassemble SNARE complexes. This proposal's Specific Aims focus on these key unanswered questions. The Aims are: 1) To determine which structural features of NSF-N are required for SNAP-SNARE complex binding and SNAP-mediated stimulation of the ATPase activity of NSF. 2) To determine what structural elements of NSF-D1 promote SNAP-dependent enhancement of nucleotide hydrolysis and facilitate the conformational changes needed for SNAP-SNARE binding and disassembly. 3) To determine the conformational changes that occur in NSF as it progresses through the different nucleotide states of its ATP hydrolysis cycle. For the first two aims, structure-based mutagenesis will be combined with a battery of functional assays to assess the importance of specific regions of NSF. For the third aim, cryo-electron microscopy and single particle image analysis will be used to generate 3D maps of the NSF hexamer in the different nucleotide-induced conformations (ADP, ATP, and ADP-Pi). These structures will then be compared to determine what parts of the NSF hexamer change and how they might shift conformations during the ATP hydrolysis cycle. From the knowledge generated by the experiments in these three specific aims, it will be possible to better understand the catalytic mechanism of NSF as well as that of a number of other cellular ATPases of the AAA family. [unreadable] [unreadable]